Use of GABAA receptor antagonists for the treatment of excessive sleepiness and disorders associated with excessive sleepiness

ABSTRACT

GABA A  receptor mediated hypersomnia can be treated by administering a GABA A  receptor antagonist (e.g., flumazenil; clarithromycin; picrotoxin; bicuculline; cicutoxin; and oenanthotoxin). In some embodiments, the GABA A  receptor antagonist is flumazenil or clarithromycin. The GABA A  receptor mediated hypersomnia includes shift work sleep disorder, obstructive sleep apnea/hypopnea syndrome, narcolepsy, excessive sleepiness, hypersomnia (e.g., idiopathic hypersomnia; recurrent hypersomnia; endozepine related recurrent stupor; and amphetamine resistant hypersomnia), and excessive sleepiness associated with shift work sleep disorder, obstructive sleep apnea/hypopnea syndrome, and hypersomnia (e.g., idiopathic hypersomnia; recurrent hypersomnia; endozepine related recurrent stupor; and amphetamine resistant hypersomnia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/922,044, filed Sep. 10, 2010, which is a filing under 35 U.S.C. § 371based on PCT/US2009/037034 filed Mar. 12, 2009, which claims priorityunder 35 U.S.C. § 119 to U.S. Provisional Application Ser. No.61/036,047, filed Mar. 12, 2008, each of which applications areincorporated by reference in their entirety herein.

TECHNICAL FIELD

The present disclosure relates to the treatment of excessive sleepinessand promotion of wakefulness in a subject. In particular, a method oftreating hypersomnia (e.g., GABA_(A) receptor mediated hypersomnia)using a GABA_(A) receptor anagonist such as flumazenil (formulated, forexample, for I.V., transdermal, transmucosal, sublingual, or subdermaladministration) is disclosed.

BACKGROUND

There are two main categories of hypersomnia: primary hypersomnia(sometimes called idiopathic hypersomnia) and recurrent hypersomnia(sometimes called idiopathic recurrent hypersomnia). Both arecharacterized by similar signs and symptoms and differ only in thefrequency and regularity with which the symptoms occur.

Primary hypersomnia is characterized by excessive daytime sleepinessover a long period of time. The symptoms are present all, or nearly all,of the time. Recurring hypersomnia involves periods of excessive daytimesleepiness that can last from one to many days, and recur over thecourse of a year or more. The primary difference between this andprimary hypersomnia is that persons experiencing recurring hypersomniawill have prolonged periods where they do not exhibit any signs ofhypersomnia, whereas persons experiencing primary hypersomnia areaffected by it nearly all the time. Idiopathic hypersomnia is much likenarcolepsy, except there is no cataplexy, no sleep paralysis, and norapid eye movement when the victim first falls asleep.

Various treatments including prescription drugs have been used to treathypersomnia without significant success, and no substantial body ofevidence supports the effectiveness of any of these treatments.Stimulants are not generally recommended to treat hypersomnia as theytreat the symptoms but not the base problem. There is a need for moreeffective treatments of hypersomnia, especially using administrationroutes that allow for better drug delivery and patient compliance.

SUMMARY

The inventors have discovered that many patients that suffer fromexcessive sleepiness or disorders associated with excessive sleepinesshave one or more endogenous substances present, typically in excess, intheir CSF that act as positive allosteric modulators of the GABA_(A)receptor, potentiating the effect of GABA on the receptor. Treatment ofsuch patients with a GABA_(A) receptor antagonist thus can provide amethod to treat the disorders, in particular the symptoms of excessivesleepiness associated with the disorders.

Accordingly, provided herein are methods of treating GABA_(A) receptormediated hypersomnia in a subject, the methods comprising administeringto the subject an effective amount of a GABA_(A) receptor antagonist. Inaddition, provided herein is a method of treating excessive sleepinessassociated with GABA_(A) receptor mediated hypersomnia in a subject,comprising administering to the subject an effective amount of aGABA_(A) receptor antagonist. In some embodiments, the GABA_(A) receptormediated hypersomnia is selected from one or more of: shift work sleepdisorder; narcolepsy; obstructive sleep apnea/hypopnea syndrome; REMbehavior disorder; frontal nocturnal dystonia; restless legs syndrome;nocturnal movement disorder; Kleine-Levin syndrome; Parkinson's disease;excessive sleepiness; hypersomnia; idiopathic hypersomnia; recurrenthypersomnia; endozepine related recurrent stupor; and amphetamineresistant hypersomnia. In some embodiments, the GABA_(A) receptormediated hypersomnia is a result of the production of endogenoussomnogenic compounds in a subject, e.g., excessive amounts of somnogeniccompounds. In some embodiments, the GABA_(A) receptor antagonist can bea negative allosteric modulator. In some embodiments, the GABA_(A)receptor antagonist is selected from the group consisting of:flumazenil; clarithromycin; picrotoxin; bicuculline; cicutoxin; andoenanthotoxin. In some embodiments, the method includes administering anI.V., transdermal, transmucosal, sublingual, or subdermal formulation ofthe GABA_(A) receptor antagonist to the subject.

Also provided herein is a method of treating excessive sleepiness in asubject. The method comprises the steps of determining whether thesubject has an endogenously produced somnogenic compound in a CSF sampleof the subject, e.g., an excessive amount of the somnogenic compound;and administering to the subject an effective amount of a GABA_(A)receptor antagonist, e.g., flumazenil. The step of determining whetherthe subject has an endogenously produced somnogenic compound, includingan excessive amount of the somnogenic compound, includes the steps of:a) measuring the potentiation of GABA_(A) receptors contacted with theCSF sample of the subject in a whole cell patch clamp assay, wherein thecells express benzodiazepine sensitive receptors; b) measuring thepotentiation of GABA_(A) receptors contacted with the CSF sample of thesubject in a whole cell patch clamp assay, wherein the cells expressbenzodiazepine insensitive receptors; and c) comparing the response ofstep a) to the response of step b), wherein a persistence ofpotentiation in step b) to within ±25% of the step a) response isindicative of an endogenously produced somnogenic compound in the CSFsample of the subject. In some embodiments, the somnogenic compound is anon-classical benzodiazepine. In some embodiments, the somnogeniccompound binds to a site on the GABA_(A) receptor, e.g., an allostericsite. In some embodiments, the site on the GABA_(A) receptor is otherthan the benzodiazepine binding site.

A method of treating excessive sleepiness of a subject endogenouslyproducing a somnogenic compound, e.g., an excessive amount of asomnogenic compound, is also provided herein, the method comprisingadministering to the subject an effective amount of a GABA_(A) receptorantagonist, e.g., flumazenil. Further described herein is a method ofdetermining whether a subject will benefit from treatment with aGABA_(A) receptor antagonist, e.g., flumazenil, wherein the benefit is areduction in excessive sleepiness, the method comprising determiningwhether the subject has an endogenously produced somnogenic compound,e.g., an excess of the somnogenic compound, in a CSF sample of thesubject, wherein the presence of the endogenously produced somnogeniccompound is indicative that the subject will benefit from treatment withGABA_(A) receptor antagonist, e.g., flumazenil. In the method, thedetermining step comprises: a) measuring the potentiation of GABA_(A)receptors contacted with the CSF sample of the subject in a whole cellpatch clamp assay, wherein the cells express benzodiazepine sensitivereceptors; b) measuring the potentiation of GABA_(A) receptors contactedwith the CSF sample of the subject in a whole cell patch clamp assay,wherein the cells express benzodiazepine insensitive receptors; and c)comparing the response of step a) to the response of step b), wherein apersistence of potentiation to within ±25% of the step a) response isindicative that the subject will benefit from treatment with flumazenil.

In some embodiments of the methods described herein, the GABA_(A)receptor antagonist is a negative allosteric modulator. In someembodiments, the GABA_(A) receptor antagonist is selected from the groupconsisting of: flumazenil; clarithromycin; picrotoxin; bicuculline;cicutoxin; and oenanthotoxin. In some embodiments, the GABA_(A) receptorantagonist is flumazenil. In some embodiments, the GABA_(A) receptorantagonist is clarithromycin.

Further provided herein are methods of treating disorders associatedwith excessive sleepiness (e.g., GABA_(A) receptor mediated hypersomnia)and symptoms of excessive sleepiness in a subject. In some embodiments,the method includes administering an I.V., transdermal, transmucosal,sublingual, or subdermal formulation of a GABA_(A) receptor antagonist,e.g., selected from flumazenil; clarithromycin; picrotoxin; bicuculline;cicutoxin; and oenanthotoxin to the subject.

A disorder associated with excessive sleepiness can be selected from oneor more of: shift work sleep disorder; narcolepsy; obstructive sleepapnea/hypopnea syndrome; hypersomnia; REM behavior disorder; frontalnocturnal dystonia; restless legs syndrome; nocturnal movement disorder;Kleine-Levin syndrome; and Parkinson's disease. In some embodiments, thedisorder is hypersomnia, for example GABA_(A) receptor mediatedhypersomnia (e.g., idiopathic hypersomnia; recurrent hypersomnia;endozepine related recurrent stupor; and amphetamine resistanthypersomnia).

A method of treating a disorder associated with excessive sleepiness ina subject is provided, the method comprising administering to thesubject an effective amount of a transmucosal, transdermal, or I.V.formulation of a GABA_(A) receptor antagonist, e.g., flumazenil. In someembodiments, treating a disorder associated with excessive sleepinesscan include administering an effective amount of a GABA_(A) receptorantagonist, e.g., flumazenil, using a subdermal pump.

In some embodiments, a transmucosal formulation of a GABA_(A) receptorantagonist, e.g., flumazenil, is administered. The transdermalformulation can be administered supralingually, sublingually, orbuccally.

In some embodiments, the subject is administered about 2 mg flumazenilper Body Mass Index unit of the subject over a 24 hour period.Administration may be self-administered by the patient as needed, or inthe case of an I.V. or subdermal route of administration, the flumazenilcan be administered automatically. In some embodiments, the effectiveamount of flumazenil is about 6 mg per dose six times per day.

Independent of the formulation and route of administration, any of themethods may further comprise administering a wakefulness promoting agent(e.g., modafinil and armodafinil). In some embodiments, the wakefulnesspromoting agent is modafinil. In some embodiments, the method comprisesadministering a time-release formulation of a GABA_(A) receptorantagonist, such as a time-release transdermal formulation.

Further provided herein is a method of treating a GABA_(A) receptormediated hypersomnia in a subject, the method comprising: a)administering to the subject a sublingual formulation of a GABA_(A)receptor antagonist, e.g., flumazenil; and b) administering to thesubject a wakefulness promoting agent. In some embodiments, the methodcomprises: a) administering flumazenil in an amount of about 2 mg offlumazenil per Body Mass Index unit of the subject per 24 hour period;and b) administering to the subject a wakefulness promoting agent. Alsoprovided is a method of treating a GABA_(A) receptor mediatedhypersomnia in a subject, the method comprising: a) administering to thesubject a GABA_(A) receptor antagonist, e.g., flumazenil, using asubdermal pump; and b) administering to the subject a wakefulnesspromoting agent. In some embodiments, a method of treating a GABA_(A)receptor mediated hypersomnia in a subject is provided, the methodcomprising: a) administering to the subject an I.V. formulation offlumazenil in an amount of about 0.2 mg to about 2 mg; and b)administering to the subject a wakefulness promoting agent. In someembodiments, the methods described above further comprise administrationof a transdermal formulation of a GABA_(A) receptor antagonist, e.g.,flumazenil.

A method of treating a disorder associated with excessive sleepiness ina subject is provided, the method comprising administering a GABA_(A)receptor antagonist, e.g., flumazenil, in an amount effective todecrease the subject's CSF-induced enhancement of whole cell patch clampassayed GABA_(A)R responses in the presence of GABA such that theresponses in the presence of GABA are within ±25% of a control sample.In some embodiments, a method of treating a disorder associated withexcessive sleepiness in a subject is provided, the method comprisingadministering a GABA_(A) receptor antagonist, e.g., flumazenil, in anamount effective to modulate the response of a CSF sample of the subjectas measured in a GABA whole cell patch clamp assay to within ±25% of theresponse of a control sample. In some embodiments, the modulation is adecrease in the response of the CSF sample of the subject in thepresence of a GABA_(A) receptor antagonist, e.g., flumazenil.

A method of testing a subject for the presence of a positive allostericmodulator of GABA_(A) receptor function in a CSF or blood sample is alsoprovided, the method comprising measuring the response of GABA_(A)Receptors contacted with the CSF or blood and with GABA in a whole cellpatch clamp assay, and comparing the response to a control sample,wherein a greater than 50% increase in the response relative to thecontrol is indicative of the presence of a positive allosteric modulatorof GABA_(A) receptor function.

Also provided herein are methods of treating shift work sleep disorder,obstructive sleep apnea/hypopnea syndrome, and narcolepsy in a subject,the methods comprising administering to the subject an effective amountof a GABA_(A) receptor antagonist, e.g., flumazenil. A method oftreating excessive sleepiness associated with shift work sleep disorder,obstructive sleep apnea/hypopnea syndrome, hypersomnia (e.g., idiopathichypersomnia; recurrent hypersomnia; endozepine related recurrent stupor;and amphetamine resistant hypersomnia), or narcolepsy in a subject isalso provided, the method comprising administering to the subject aneffective amount of a GABA_(A) receptor antagonist, e.g., flumazenil. Insome embodiments, the a GABA_(A) receptor antagonist is an I.V.formulation, a transdermal formulation, or a transmucosal formulation.

A method of altering a somnolent state of a subject is further providedherein, the method comprising administering to the subject an effectiveamount of a GABA_(A) receptor antagonist, e.g., flumazenil. Thesomnolent state is selected from one or more of: narcolepsy, obstructivesleep apnea/hypopnea syndrome, shift work sleep disorder, andhypersomnia (e.g., idiopathic hypersomnia; recurrent hypersomnia;endozepine related recurrent stupor; and amphetamine resistanthypersomnia). In some embodiments, the a GABA_(A) receptor antagonist isan I.V. formulation, a transdermal formulation, or a transmucosalformulation.

Also provided herein are methods for enhancing alertness or increasingregularity of sleep rhythms in a subject; promoting wakefulness in asubject; improving cognitive dysfunction in a subject; and restoring anormal sleep pattern and improving the quality of psychosocial life andrelationships in a subject, each method comprising administering to thesubject an effective amount of a GABA_(A) receptor antagonist, e.g.,flumazenil. In some embodiments, the a GABA_(A) receptor antagonist isan I.V. formulation, a transdermal formulation, or a transmucosalformulation.

A method of characterizing the phenotypic spectrum of GABA_(A) receptormediated hypersomnia is also provided, the method comprising measuringthe potentiation of GABA_(A) receptor function of a CSF or plasma sampleof at least one subject having a disorder associated with excessivesleepiness, and correlating the potentiation with at least one measureof sleep or sleepiness of the subject, wherein a positive correlation isindicative that the subject's disorder is within the phenotypic spectrumof a GABA_(A) receptor mediated hypersomnia. In some embodiments, themeasure of sleep and sleepiness is a behavioral assessment, anelectroencephalographic assessment, or a subjective assessment. Themethod can further comprise quantifying GABA_(A) receptor function.

Further provided herein are uses of a GABA_(A) receptor antagonist suchas flumazenil for the manufacture of medicaments for the treatment ofthe following disorders and conditions: obstructive sleep apnea/hypopneasyndrome; shift work sleep disorder; narcolepsy; hypersomnia; andexcessive sleepiness associated with shift work sleep disorder,obstructive sleep apnea/hypopnea syndrome, hypersomnia, or narcolepsy.In some embodiments, the hypersomnia is selected from one or more of:idiopathic hypersomnia; recurrent hypersomnia; endozepine relatedrecurrent stupor; and amphetamine resistant hypersomnia.

Also provided herein are uses of a GABA_(A) receptor antagonist such asflumazenil for the manufacture of medicaments for altering a somnolentstate of a subject; enhancing alertness or increasing regularity ofsleep rhythms in a subject; promoting wakefulness in a subject;improving cognitive dysfunction in a subject; and restoring a normalsleep pattern and improving the quality of psychosocial life andrelationships in a subject. In some embodiments, the somnolent state isselected from one or more of: narcolepsy; obstructive sleepapnea/hypopnea syndrome; shift work sleep disorder; and hypersomnia. Insome embodiments, the hypersomnia is selected from one or more of:idiopathic hypersomnia; recurrent hypersomnia; endozepine relatedrecurrent stupor; and amphetamine resistant hypersomnia. In someembodiments, a GABA_(A) receptor antagonist such as flumazenil isformulated for administration by a transdermal, transmucosal, orintravenous route for the uses described herein.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates whole cell patch clamp recordings of GABA_(A)Rfunction with and without flumazenil.

FIG. 2 shows that human α1β2γ2s GABA_(A) receptor function is enhancedby the plasma of a subject suffering from hypersomnia. Enhancement isreduced following administration of 12 mg of a sublingual formulation offlumazenil.

FIG. 3 is a graph illustrating that potentiation of GABA_(A) function isevident in human controls absent sleep related complaints, non-human(rhesus) primates, and in excess in many hypersomnic patients.

FIG. 4 shows the power spectrum analyses results obtained fromprocessing 27 minutes of non-artifactual data for subject DS122 afterinfusion with 2.0 mg flumazenil.

FIG. 5 shows the power spectrum analyses results obtained fromprocessing 19 minutes of non-artifactual data for subject DT74 afterinfusion with 2.0 mg flumazenil.

FIG. 6 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance before administration of I.V.flumazenil for case 74.

FIG. 7 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance after 2.0 mg dose of I.V. flumazenilfor case 74.

FIG. 8 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance before administration of I.V.flumazenil for case 102.

FIG. 9 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance after 2.0 mg dose of I.V. flumazenilfor case 102.

FIG. 10 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance before administration of I.V.flumazenil for case 122.

FIG. 11 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance after 2.0 mg dose of I.V. flumazenilfor case 122.

FIG. 12 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance before administration of I.V.flumazenil for case 124.

FIG. 13 shows a histogram displaying the results of the psychomotorvigilance task (PVT) performance after 1.2 mg dose of I.V. flumazenilfor case 124.

FIG. 14 shows a graph displaying the results of the psychomotorvigilance task (PVT) performance before and after treatment with I.V.flumazenil for case 74.

FIG. 15 shows a graph displaying the results of the psychomotorvigilance task (PVT) performance before and after treatment with I.V.flumazenil for case 102.

FIG. 16 shows a graph displaying the results of the psychomotorvigilance task (PVT) performance before and after treatment with I.V.flumazenil for case 122.

FIG. 17 shows a graph displaying the results of the psychomotorvigilance task (PVT) performance before and after treatment with I.V.flumazenil for case 124.

FIG. 18a is an illustration of the rest-activity cycle of patient AS99before treatment with flumazenil.

FIG. 18b is an illustration of the rest-activity cycle of patient AS99after treatment with flumazenil.

FIG. 19a illustrates a whole cell patch clamp recording from a cellexpressing human α1β2γ2s receptors. Bars above the traces indicateduration of GABA and CSF application.

FIG. 19b illustrates a whole cell patch clamp recording from a cellexpressing the benzodiazepine insensitive subunit α1(H102R). Bars abovethe traces indicate duration of GABA and CSF application.

DETAILED DESCRIPTION

Ionotropic GABA_(A) receptors (GABA_(A)R) are the most recognizedtherapeutic targets for anesthetics and sedative/hypnotic drugs.Mutations in the α1, γ2, and delta subunits of GABA_(A) R account forseveral of the heritable epilepsies, endogenous positive allostericneurosteroid modulators contribute to fluctuations in mood due todevelopmental changes in expression of the α4δ. GABA_(A)R, and mutationof the β3 subunit has been associated with chronic insomnia. Theinventors have found that a naturally occurring endogenous, positive,allosteric modulator of recombinant α1, β2, γ2 short splice variantGABA_(A)R is present in CSF plasma in normal humans and non-humanprimates, and when present in excess, produces hypersomnia and excessivedaytime sleepiness, or GABA_(A) receptor mediated hypersomnia (GRH) asdescribed herein. Accordingly, treatment of such patients with aGABA_(A) receptor antagonist thus can provide a method to treat patientshaving various disorders associated with excessive sleepiness, and inparticular treat the symptoms of excessive sleepiness associated withthe various disorders.

I. Methods of Treating GABA_(A) Receptor Mediated Hypersomnia andDisorders Associated with Excessive Sleepiness

Provided herein are methods of treating GABA_(A) receptor mediatedhypersomnia in a subject, the methods comprising administering to thesubject an effective amount of a GABA_(A) receptor antagonist. Inaddition, provided herein is a method of treating excessive sleepinessassociated with GABA_(A) receptor mediated hypersomnia in a subject,comprising administering to the subject an effective amount of aGABA_(A) receptor antagonist. In some embodiments of the methodsdescribed herein, the GABA_(A) receptor antagonist is a negativeallosteric modulator. In some embodiments, the GABA_(A) receptorantagonist is selected from the group consisting of: flumazenil;clarithromycin; picrotoxin; bicuculline; cicutoxin; and oenanthotoxin.In some embodiments, the GABA_(A) receptor antagonist is flumazenil. Insome embodiments, the GABA_(A) receptor antagonist is clarithromycin. Insome embodiments, the method includes administering a I.V., transdermal,transmucosal, sublingual, or subdermal formulation of flumazenil to thesubject. The administration of flumazenil can be combined withadministration of other agents, including wakefulness promoting agentsand transdermal formulations of flumazenil.

GABA_(A) receptor mediated hypersomnia or disorders associated withexcessive sleepiness are selected from one or more of: shift work sleepdisorder; narcolepsy; obstructive sleep apnea/hypopnea syndrome; REMbehavior disorder; frontal nocturnal dystonia; restless legs syndrome;nocturnal movement disorder; Kleine-Levin syndrome; Parkinson's disease;excessive sleepiness; hypersomnia; idiopathic hypersomnia; recurrenthypersomnia; endozepine related recurrent stupor; and amphetamineresistant hypersomnia. In some embodiments, the GABA_(A) receptormediated hypersomnia is selected from idiopathic hypersomnia; recurrenthypersomnia; endozepine related recurrent stupor; and amphetamineresistant hypersomnia. In some embodiments, the hypersomnia isidiopathic hypersomnia. In some embodiments, the hypersomnia isendozepine related recurrent stupor. In some embodiments, thehypersomnia is amphetamine resistant hypersomnia.

Such disorders can be characterized by many objective and subjectivetests known in the art. For example, the Epworth Sleepiness Scale; theStanford Sleepiness Scale; the Pittsburgh Sleep Quality Index; anActivity-Rest and Symptom Diary; Actigraphy; Psychomotor Vigilance Task;Polysomnography; Functional Magnetic Resonance Imaging; Profile of MoodStates; Functional Outcomes of Sleep Questionnaire; Medical OutcomesStudy Short-Form 36; and Neurophysical Testing, such as the CambridgeNeurophysical Test Automated Battery (CANTAB) (e.g., physcomotor speed,attention, working memory, and executive function).

In addition, GABA_(A) receptor mediated hypersomnia can be characterizedby demonstration of enhanced GABA_(A) Receptor function of a subject'sCSF or plasma as compared to a control, e.g., see Example 1 and Example14.

II. Methods of Promoting Wakefulness and Enhancing Alertness inSleepiness Associated Disorders

Further provided herein are methods of treating GABA_(A) mediatedhypersomnia disorders, including shift work sleep disorder, obstructivesleep apnea/hypopnea syndrome, narcolepsy, and excessive sleepinessassociated with shift work sleep disorder, obstructive sleepapnea/hypopnea syndrome, hypersomnia, and narcolepsy. In someembodiments, the GABA_(A) mediated hypersomnia is idiopathichypersomnia; recurrent hypersomnia; endozepine related recurrent stupor;or amphetamine resistant hypersomnia. The method comprises administeringto the subject an effective amount of a GABA_(A) receptor antagonist,such as flumazenil. In some embodiments, the GABA_(A) receptorantagonist is an I.V. formulation, a transdermal formulation, or atransmucosal formulation.

A method of altering a somnolent state of a subject is further providedherein, the method comprising administering to the subject an effectiveamount of GABA_(A) receptor antagonist, e.g., flumazenil. The somnolentstate is selected from one or more of: narcolepsy, obstructive sleepapnea/hypopnea syndrome, shift work sleep disorder, and hypersomnia(e.g., idiopathic hypersomnia; recurrent hypersomnia; endozepine relatedrecurrent stupor; and amphetamine resistant hypersomnia). In someembodiments, the GABA_(A) receptor antagonist is an I.V. formulation, atransdermal formulation, or a transmucosal formulation.

Also provided herein are methods for enhancing alertness or increasingregularity of sleep rhythms in a subject; promoting wakefulness in asubject; improving cognitive dysfunction in a subject; and restoring anormal sleep pattern and improving the quality of psychosocial life andrelationships in a subject, each method comprising administering to thesubject an effective amount of GABA_(A) receptor antagonist, e.g.,flumazenil. In some embodiments, the GABA_(A) receptor antagonist is anI.V. formulation, a transdermal formulation, or a transmucosalformulation.

As used herein, the term “promoting wakefulness” refers to a decrease insleepiness, tendency to fall asleep, or other symptoms of undesired orreduced alertness or consciousness compared with sleepiness, tendency tofall asleep, or other symptoms of undesired or reduced alertness orconsciousness expected or observed without treatment. Promotingwakefulness refers to a decrease in any stage of sleep, including lightsleep, deeper sleep characterized by the presence of high amplitude, lowwave brain activity termed “slow wave sleep”, and rapid eye movement(REM) sleep.

A determination of whether the treatment is useful in performing themethods described herein can be made, for example, by direct observationof behavioral or physiological properties of mammalian sleep, byself-reporting, or by various well-known methods, includingelectrophysiological methods. Such methods include, for example,examining electroencephalograph (EEG) activity amplitude and frequencypatterns, examining electromyogram activity, and examining the amount oftime during a measurement time period, in which a mammal is awake orexhibits a behavioral or physiological property characteristic ofwakefulness.

The effectiveness of the treatments can also be characterized by theobjective and subjective tests described herein, including the EpworthSleepiness Scale; the Stanford Sleepiness Scale; the Pittsburgh SleepQuality Index; an Activity-Rest and Symptom Diary; Actigraphy;Psychomotor Vigilance Task; Polysomnography; Functional MagneticResonance Imaging; Profile of Mood States; Functional Outcomes of SleepQuestionnaire; Medical Outcomes Study Short-Form 36; and NeurophysicalTesting, such as the Cambridge Neurophysical Test Automated Battery(CANTAB) (e.g., physcomotor speed, attention, working memory, andexecutive function).

III. Formulation and Administration of a GABA_(A) Receptor Antagonist

A GABA_(A) receptor antagonist can be selected from flumazenil;clarithromycin; picrotoxin; bicuculline; cicutoxin; and oenanthotoxinand can be formulated for I.V., transdermal, transmucosal, sublingual,oral, and subdermal administration for use with the methods describedherein. A transmucosal formulation can include sublingual, supralingual,and buccal administration. For transmucosal administration, theantagonist may be combined with one or more inactive ingredients for thepreparation of a tablet, packed powder, edible film strip, soft gelcapsule, hard gel capsule, lozenge, or troches. For example, in someembodiments, the antagonists such as flumazenil may be combined with atleast one excipient such as fillers, binders, humectants, disintegratingagents, solution retarders, absorption accelerators, wetting agentsabsorbents, or lubricating agents. According to some embodiments, theantagonist may be combined with one or more of a polyol (e.g., lactose,sucrose, mannitol, or mixtures thereof), an alcohol (e.g., ethanol), anda gum (e.g., acacia and guar), and then formed into a lozenge byconventional methods.

In some embodiments, the formulation is a hard, compressed, rapidlydissolving tablet adapted for direct sublingual dosing. The tabletincludes particles made of the antagonist and a protective material. Insome embodiments, these particles are provided in an amount of betweenabout 0.01 and about 75% by weight based on the weight of the tablet(e.g., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 60%, 70%, and75%). In some embodiments, the tablet may also include a matrix madefrom a nondirect compression filler, a wicking agent, and a hydrophobiclubricant. In some embodiments, the tablet is adapted to dissolvespontaneously in the mouth of a patient in less than about 60 seconds(and, in some cases, in less than about 30 seconds).

In some embodiments, the formulation can be a compressed rapidlydissolving tablet comprising effervescent agents. These effervescentagents allow enhanced adsorption of the antagonist across the mucosalmembranes (e.g., tongue, cheek, and gums) in the oral cavity. An exampleof effervescent pharmaceutical compositions suitable for use inconjunction with the methods described herein are the compositionsdescribed in U.S. Pat. No. 6,200,604.

In some embodiments, the antagonist can be administered transmucosallyusing an edible film. Such films can include a carrier comprisingwater-soluble polymers in combination with certain ingredients andprovides a therapeutic effect. In some embodiments, the film is coatedand dried utilizing existing coating technology and exhibits instantwettability followed by rapid dissolution/disintegration uponadministration in the oral cavity. In some embodiments, an edible filmcan contain as the essential components a water-soluble polymer or acombination of water-soluble polymers, one or more plasticizers orsurfactants, one or more polyalcohols, and flumazenil. Non-limitingexamples of edible films can be found in U.S. Pat. Nos. 5,948,430;6,177,096; 6,284,264; 6,592,887; and 6,709671.

Further examples of additional pharmaceutical compositions suitable fortransmucosal administration include those described in U.S. Pat. Nos.5,178,878; 5,223,264; and 6,024,981.

In some embodiments, the antagonist is combined with inactiveingredients. Such ingredients may be necessary, for example, to add bulkto the pharmaceutical preparation, to bind the preparation, to add coloror flavor to the preparation, and to prevent degradation or growth ofcontaminants.

In some embodiments, administration of the antagonist may be performedusing an implantable device, for example, an implantable,self-regulating mechanochemical subdermal pump. In some embodiments, thedevice may administer the antagonist on a set dosage program. In someembodiments, the device may administer the antagonist on demand asdetermined by the subject. In some embodiments, the device mayadminister the antagonist on a constant release profile. In someembodiments, the device may administer the antagonist automatically.These devices are known in the art for the treatment of other disorders,for example, diabetes. Non-limiting examples of various embodiments ofthis mode of administration are detailed in U.S. Pat. Nos. 5,062,841;5,324,518; and 6,852,104.

In some embodiments, a transmucosal administration of an antagonist maybe combined with transdermal administration of the same or anotherantagonist. Without being bound by theory, such a delivery mechanism maybe useful for nocturnal application to assist the subject with morningwakefulness.

Transdermal administration of the antagonist can be accomplished bymixing the antagonist with suitable pharmaceutical carriers,preservatives, optional penetration enhancers, and optional gellingagents to form ointments, emulsions, lotions, solutions, creams, gels,patches or the like, wherein a fixed amount of the preparation isapplied onto a certain area of skin.

By the term “suitable pharmaceutical carrier” is meant a non-toxicpharmaceutically acceptable vehicle including, for example, polyethyleneglycol, propylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, sesame oil, olive oil, wood alcohol ointments,vaseline, and paraffin or a mixture thereof.

Suitable penetration enhancers include, for example, saturated andunsaturated fatty acids and their esters, alcohols, monoglycerides,diethanolamines, N,N-dimethylamines such as linolenic acid, linolenylalcohol, oleic acid, oleyl alcohol, stearic acid, stearyl alcohol,palmitic acid, palmityl alcohol, myristic acid, myristyl alcohol,1-dodecanol, 2-dodecanol, lauric acid, decanol, capric acid, octanol,caprylic acid, 1-dodecylazacycloheptan-2-one sold under the trademarkAZONE (Nelson Research and Development; Irvine, Calif.), ethylcaprylate, isopropyl myristate, hexamethylene lauramide, hexamethylenepalmitate, capryl alcohol, decyl methyl sulfoxide, dimethyl sulfoxide,salicylic acid and its derivatives, N,N-diethyl-m-toluamide, crotamiton,1-substituted azacycloalkan-2-ones, polyethylene glycol monolaurate andany other compounds compatible with medetomidine and its opticallyactive enantiomers and the packages and having transdermal permeationenhancing activity.

Suitable gelling agents include, for example, hydroxy methyl cellulose,hydroxypropyl cellulose sold under the trademark KLUCEL HF (HerculesInc.; Wilmington, Del.), tragacanth, sodium alginate, gelatin,methylcellulose, sodium carboxymethylcellulose, and polyvinyl alcohols.Suitable preservatives include, for example, parabens, benzoic acid, andchlorocresol.

Antioxidants can be included in the formulations described herein.Suitable antioxidants include, for example, ascorbyl palmirate,butylated hydroxyanisole, butylated hydroxytoluene, potassium sorbate,sodium bisulfate, sorbic acid, propyl gallate, and sodium metabisulfite.

In some embodiments, the antagonist is administered by a transdermalpatch. Adhesives for making transdermal patches for use in the methodsdescribed herein include polyisobutylene, silicone based adhesives, andacrylic polymers. The adhesive polymers can be mixed with otherexcipients such as waxes and oils (e.g., mineral oil). A protectiveliner can be placed in contact with the adhesive layer to protectagainst drug release from the patch prior to application. Liners for usewith the transdermal patches described herein include, for example,polyethylene terephthalate film, polyester membrane, and polycarbonatefilm.

The backing membrane of the transdermal patch for use with the methodsdescribed herein constitutes the top face surface of the transdermalpatch. It may be made of a single layer or film of polymer, or be alaminate of one or more polymer layers and metal foil. Examples ofpolymers suitable for use in making backing films include, for example,polyester films, ethyl vinyl acetate, polypropylene, polyethylene, andpolyvinyl-chloride.

In some embodiments, the administration rate of the drug is 0.1-1000μg/h through a skin area of about 2-90 cm² (e.g., 10-30 cm²). The amountof drug delivered into the skin can be controlled by a number of factorsincluding skin patch size, degree of drug loading, the use of ratecontrolling membranes, permeation enhancers, and the like.

In some embodiments, the transmucosal and/or the transdermal formulationmay be a time-release or slow-release formulation. In some embodiments,the transdermal formulation may be a time-release or slow-releaseformulation. The transmucosal or transdermal formulation describedherein may also be formulated so as to provide slow or controlledrelease of the antagonist using, for example, hydropropylmethylcellulose in varying proportions to provide the desired release profile,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes and/or microspheres. Ingeneral, a controlled-release preparation is a pharmaceuticalcomposition capable of releasing the active ingredient at the requiredrate to maintain constant pharmacological activity for a desirableperiod of time. Such dosage forms provide a supply of a drug to the bodyduring a predetermined period of time and thus maintain drug levels inthe therapeutic range for longer periods of time than conventionalnon-controlled formulations.

U.S. Pat. No. 5,591,767 describes a liquid reservoir transdermal patchfor the controlled administration of ketorolac, a non-steroidalanti-inflammatory agent with potent analgesic properties. U.S. Pat. No.5,120,548 discloses a controlled-release drug delivery device comprisedof swellable polymers. U.S. Pat. No. 5,073,543 describescontrolled-release formulations containing a trophic factor entrapped bya ganglioside-liposome vehicle. U.S. Pat. No. 5,639,476 discloses astable solid controlled-release formulation having a coating derivedfrom an aqueous dispersion of a hydrophobic acrylic polymer.Biodegradable microparticles are known for use in controlled-releaseformulations. U.S. Pat. No. 5,354,566 discloses a controlled-releasepowder that contains the active ingredient. U.S. Pat. No. 5,733,566describes the use of polymeric microparticles that release antiparasiticcompositions.

The controlled-release of the active ingredient may be stimulated byvarious inducers, for example, pH, temperature, enzymes, water, or otherphysiological conditions or compounds. Various mechanisms of drugrelease exist. For example, in one embodiment, the controlled-releasecomponent may swell and form porous openings large enough to release theantagonist after administration to a patient. The term“controlled-release component” means a compound or compounds, such aspolymers, polymer matrices, gels, permeable membranes, liposomes and/ormicrospheres that facilitate the controlled-release of the activeingredient in the pharmaceutical composition. In another embodiment, thecontrolled-release component is biodegradable, induced by exposure tothe aqueous environment, pH, temperature, or enzymes in the body.

The specific dose of an antagonist required to obtain therapeuticbenefit in the methods of treatment described herein will, usually bedetermined by the particular circumstances of the individual patientincluding the size, weight, age, and sex of the subject, the nature andstage of the disorder being treated, the aggressiveness of the disorder,and the route of administration of the compound.

For transmucosal administration (e.g., sublingual administration), forexample, a daily dosage of flumazenil, for example, can range from about0.5 mg to about 10 mg per Body Mass Index (BMI) unit (e.g., about 0.5 mgto about 5 mg; about 1 mg to about 3 mg; about 1.5 mg to about 4 mg;about 2 mg to about 6 mg; about 1.25 mg to about 8 mg; and about 4 mg toabout 10 mg). In some embodiments, a daily dosage of flumazenil canrange from about 1 mg per BMI to about 5 mg per BMI. In someembodiments, a daily dosage of flumazenil can be about 1.5 mg per BMI.In some embodiments, a daily dosage of flumazenil can be about 2 mg perBMI unit. In some embodiments, a daily dosage of flumazenil can be about3 mg per BMI unit. For example, a subject with a BMI of 20 could beadministered a daily dosage of about 40 mg of flumazenil, in otherwords, a daily dosage of 2 mg per BMI unit. Higher or lower doses arealso contemplated, as it may be necessary to use dosages outside theseranges in some cases.

The transmucosal formulation can be administered in one single dosage orthe daily dosage may be divided, such as being divided equally into twoto six times per day daily dosing. In some embodiments, the transmucosalformulation is administered at least twice daily. In some embodiments,the transmucosal formulation is administered at least three times daily.In some embodiments, the transmucosal formulation is administered aboutevery one to six hours (e.g., about every one hour; about every twohours; about every three hours; about every three and a half hours;about every four hours; about every five hours; and about every sixhours). In some embodiments, the transmucosal formulation isadministered by the subject as needed, e.g., patient controlledtitration to a desired end effect (e.g., wakefulness or reducedsleepiness).

A transmucosal formulation may be formulated in a unit dosage form, eachdosage containing from about 0.5 to about 20 mg of the antagonist, e.g.,flumazenil, per unit dosage (e.g., about 0.5 mg to about 15 mg; about 1mg to about 10 mg; about 1.5 mg to about 8 mg; about 2 mg to about 7 mg;about 3 mg to about 6 mg; about 4 mg to about 8 mg; about 5 mg to about10 mg; about 6 mg to about 12 mg; and about 8 mg to about 20 mg). Insome embodiments, each dosage can contain about 5 to about 10 mg of theantagonist per unit dosage. In some embodiments, each dosage containsabout 6 mg of the antagonist. The term “unit dosage form” refers tophysically discrete units suitable as a unitary dosage for humansubjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

For transdermal administration, for example, a daily dosage offlumazenil can range from about 0.5 mg to about 10 mg (e.g., about 0.5mg to about 5 mg; about 1 mg to about 3 mg; about 1.5 mg to about 4 mg;about 2 mg to about 6 mg; about 1.25 mg to about 8 mg; and about 4 mg toabout 10 mg). In some embodiments, a daily dosage of transdermalflumazenil can range from about 1 mg to about 5 mg. In some embodiments,a daily dosage of transdermal flumazenil can be about 1.5 mg. In someembodiments, a daily dosage of transdermal flumazenil can be about 2 mg.In some embodiments, a daily dosage of transdermal flumazenil can beabout 3 mg. Higher or lower doses are also contemplated as it may benecessary to use dosages outside these ranges in some cases.

The transdermal formulation can be administered in one single dosage orthe daily dosage may be divided, such as being divided equally into twoto six times per day daily dosing. In some embodiments the transdermalformulation is formulated to a concentration of about 0.5 mg to about 10mg per mL (e.g., about 0.5 mg to about 8 mg per mL; about 1 mg to about6 mg per mL; about 1.5 mg to about 5 mg per mL; about 3 mg to about 7 mgper mL; about 4 mg to about 10 mg per mL; and about 4 mg to about 8 mgper mL). In some embodiments, the transdermal formulation is formulatedto a concentration of about 4 mg per mL. In some embodiments, thetransdermal formulation is administered once daily (e.g., before bed).In some embodiments, the transdermal formulation is administered atleast twice daily. In some embodiments, the transdermal formulation isadministered about every eight to about twenty-four hours (e.g., aboutevery eight hours; about every ten hours; about every twelve hours;about every sixteen hours; about every twenty hours; about everytwenty-two hours; and about every twenty-four hours).

A transdermal formulation may be formulated in a unit dosage form, eachdosage containing from about 0.5 to about 10 mg of flumazenil per unitdosage (e.g., about 0.5 mg to about 8 mg; about 1 mg to about 5 mg;about 1.5 mg to about 4 mg; about 2 mg to about 6 mg; about 3 mg toabout 7 mg; about 4 mg to about 8 mg; and about 5 mg to about 10 mg). Insome embodiments, each dosage can contain about 1 to about 4 mg offlumazenil per unit dosage. In some embodiments, each dosage containsabout 2 mg of flumazenil. The term “unit dosage form” refers tophysically discrete units suitable as a unitary dosage for humansubjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The components used to formulate the pharmaceutical compositionsdescribed above are of high purity and are substantially free ofpotentially harmful contaminants (e.g., at least National Food grade,generally at least analytical grade, and more typically at leastpharmaceutical grade). Particularly for human consumption, thecomposition is preferably manufactured or formulated under GoodManufacturing Practice standards as defined in the applicableregulations of the U.S. Food and Drug Administration. For example,suitable formulations may be sterile and/or substantially isotonicand/or in full compliance with all Good Manufacturing Practiceregulations of the U.S. Food and Drug Administration.

The antagonist can be administered in combination with other agents. Inone embodiment, the antagonist is administered with a wakefulnesspromoting agent (e.g., modafinil and armodafinil). In some embodiments,the wakefulness promoting agent is modafinil. In some embodiments, thesubject may be resistant to one or more wakefulness promoting agentsprior to administration of the antagonist. The wakefulness promotingagent can be administered in an amount less than about 600 mg per day(e.g., less than about 100 mg per day; less than about 200 mg per day;less than about 300 mg per day; less than about 400 mg per day; lessthan about 500 mg per day; and less than about 600 mg per day). Thespecific dose of a wakefulness promoting agent required to obtaintherapeutic benefit in the methods of treatment described herein willusually be determined by the particular circumstances of the individualsubject including the size, weight, age, and sex of the subject, thenature and stage of the disorder being treated, the aggressiveness ofthe disorder, and the route of administration of the compound. In someembodiments, the wakefulness promoting agent can be administered twicedaily. In some embodiments, the wakefulness promoting agent can beadministered in an amount of 5 mg per BMI unit. In some embodiments, thewakefulness promoting agent can be administered in an amount of 100 mgper dose. In some embodiments, the subject exhibits resistance to awakefulness promoting agent prior to administration of the antagonist.In some embodiments, administration of the antagonist can reverse ordecrease a subjects resistance to a wakefulness promoting agent.

In some embodiments, treatment of a disorder associated with excessivesleepiness can include the following:

a) transmucosal, e.g., sublingual, administration of an antagonist,e.g., flumazenil; and

b) administration of a wakefulness promoting agent.

In some embodiments, the treatment can further include:

c) transdermal administration of an antagonist, e.g., flumazenil.

For example, in some embodiments, a sublingual formulation of flumazenilis administered about every 2 to 4 hours during the waking hours of theday (e.g., every about 3 to 3.5 hours). In some embodiments, awakefulness promoting agent is administered from one to three timesduring the waking hours of the day (e.g., about every 4 hours). In someembodiments, the wakefulness promoting agent is modafinil. In someembodiments, a transdermal or time-release formulation of flumazenil isadministered once daily (e.g., before bed).

IV. Assay for GABA_(A) Receptor Mediated Hypersomnia

The GABA_(A) receptors are one of several classes of chemically gatedion channels that incorporate the features of both “receptors” and “ionchannels” into one membrane protein. These chemically gated channels(ligand gated ion channel: LGIC) can detect extracellular chemicalsignals such as neurotransmitters released from neighboring cells and inresponse will open an ion channel to allow specific ions to enter orleave the cell. When this results in a net movement of positive chargeinto the cell, the cell becomes more electrically positive and thus moreexcitable. Conversely, when this results in a net flow of negative ionsinto the cell, the neuron becomes more electrically negative and thusmore inhibited. In this way, LGICs act as chemical-to-voltage convertersand are fundamental to cell-to-cell communication and neuronal activity.Drugs and chemicals that enhance or block these functions have profoundeffect on brain circuits and ultimately human behavior. For example,most general anesthetics render patients unconscious by enhancing thefunction of inhibitory LGICs, the most common of which is the GABA_(A)receptor.

The most common inhibitory neurotransmitter in the human nervous systemis γ-aminobutyric acid or GABA. It is released by neurons at synapses,the specialized junctions between 2 neurons that permits rapidcell-to-cell communication. After leaving the presynaptic neuron andcrossing the synaptic gap, the molecules of GABA arrive at thepostsynaptic membrane where they can interact with a LGIC, the Type-AGABA receptor (GABA_(A)R). After GABA binds to the receptor, the LGICchanges shape and allows the flow of negatively charged chloride ionsinto the neuron, which results in the neuron becoming inhibited andunable to pass a message onto another neuron, until GABA unbinds and theinhibition passes.

If GABA_(A)R function is blocked, then the brain circuits in which theyare imbedded experience less inhibition. This can cause the circuits tobecome hyper-excitable, exhibiting much more excitation than normal.This will result in convulsions and seizures if the block is notremoved. This can occur in the presence of a GABA_(A)R channel blockertoxin or a GABA antagonist. This can also occur in some patients whohave inherited forms of epilepsy. In these patients, a GABA_(A)R genehas mutated to make a dysfunctional GABA_(A)R that does not function aswell as it should.

GABA_(A)RS are enhanced by many chemicals and drugs. Generalanesthetics, as already noted, enhance inhibition by making the channelsstay open for longer periods of time, increasing the duration ofinhibition. This is also true for many neurosteroids (e.g., progesteronemetabolites) and for ethanol. GABA_(A)RS are also a critical bindingsite for benzodiazepines, such as valium. These important anxiolytic andsedative drugs cause the receptors to bind GABA more tightly, alsoenhancing inhibition by the receptor.

It is important to note that all of these compounds do not activate thechannel. They are all “allosteric modulators”. They bind to sitesseparate from the GABA binding sites and simply enhance or amplify theeffect of GABA. In the absence of GABA, physiologic and/or therapeuticconcentrations of these different compounds have no effect on thechannel. GABA must be present for them to have an effect. Similarly, thebenzodiazepine antagonist flumazenil is not a GABA_(A)R blocker. Itoccupies the benzodiazepine binding site, thus blocking drugs likevalium from acting on the channel. Although it is bound to the receptor,flumazenil does not have an effect on the channel. Its functional effectcan only be observed when both GABA and a benzodiazepine are present.

Developed in the late 1970s, the single cell electrophysiology methodknown as patch clamp is a standard for measuring the function of ionchannels in research laboratories. The techniques takes advantage of thehigh electrical resistance between a cell surface and speciallyconstructed microelectrodes, and capacitative feedback electronics whichcombine to give ultra low noise (<100 fA) recordings of ions flowingthrough single ion channels.

Provided herein is a method of diagnosing and treating a patientsuffering from hypersomnia associated with the endogenous production ofGABA_(A) receptor modulators, e.g., excessive production of suchmodulators. There are many reports of hypersomnia disorders in subjectswho do not respond well to conventional stimulant (e.g., amphetamine)therapies. These subjects may be suffering from a form of hypersomniareferred to as amphetamine resistant hypersomnia, from an increasedproduction of endozepines (e.g., hemin and protoporphyrin IX), or froman increased production of another substance that binds to the GABA_(A)receptor. Without being bound by theory, the subject may be producingendogenous benzodiazepines (i.e. “endozepines”) or other somnogeniccompound(s) that interact directly or indirectly with the benzodiazepinebinding site on the GABA_(A)R, enhancing receptor function as classicbenzodiazepines such as valium.

A method of diagnosing a patient suffering from GABA_(A) mediatedhypersomnia associated with increased production of endozepines or othersomnogenic substance(s) can be performed by measuring the effect of asubjects' cerebral spinal fluid (CSF) or blood or plasma on recombinantGABA_(A)R function under whole cell patch clamp conditions (see, e.g.,FIG. 1 and Example 1 or FIGS. 19A and 19B and Example 14). In someembodiments, the effect of the CSF or blood or plasma can be compared tothe effect observed when the CSF or blood or plasma is co-applied with aGABA_(A) receptor antagonist such as flumazenil. In some embodiments,application of the antagonist such as flumazenil can modulate theresponse of a CSF or blood sample of a subject as measured in a GABAwhole cell patch clamp efficacy assay to within 25% of a control sampleresponse. In some embodiments, the modulation is a decrease in theresponse of the CSF sample of the subject in the presence of theantagonist such as flumazenil. In some embodiments, the effect of theCSF or blood or plasma in an assay expressing benzodiazepine sensitivereceptors can be compared to the effect observed of the CSF or blood orplasma in an assay expressing benzodiazepine insensitive receptors. Insome embodiments, the substance in the CSF or blood or plasma sample ofa subject potentiates the response of GABA as measured in a GABA wholecell patch clamp efficacy assay. In some embodiments, the potentiationof the GABA response in the benzodiazepine sensitive receptors and thepotentiation of the GABA response in the benzodiazepine insensitivereceptors are within ±25% of each other. In some embodiments, thepersistence of potentiation within ±25% of the GABA responses inbenzodiazepine sensitive and insensitive receptor assays is indicativethat the subject would benefit from treatment with a GABA_(A) receptorantagonist. In some embodiments, the GABA_(A) receptor antagonist isflumazenil.

Further, a method of diagnosing a patient suffering from GABA_(A)mediated hypersomnia associated with increased production of endozepinesor other somnogenic substances can be performed by measuring the effectof a subjects' cerebral spinal fluid (CSF) or blood or plasma onrecombinant GABA_(A)R function under whole cell patch clamp conditions.

V. Kits

Also provided herein are kits for treating disorders associated withexcessive sleepiness. A kit can include an I.V., transdermal, oral, ortransmucosal (e.g., sublingual, supralingual, and buccal) formulation ofa GABA_(A) receptor antagonist. In some embodiments, the GABA_(A)receptor antagonist is flumazenil. In some embodiments, the kit canfurther includes one or more of a wakefulness promoting agent (e.g.,modafinil) and a transdermal formulation of a GABA_(A) receptorantagonist. In some embodiments, a kit can include one or more deliverysystems and directions for use of the kit (e.g., instructions fortreating a subject). In some embodiments, a kit can include a sublingualformulation of flumazenil and a transdermal formulation of flumazenil.In another embodiment, a kit can include a sublingual formulation offlumazenil and a wakefulness promoting agent. In some embodiments, thekit can include a sublingual formulation of flumazenil and a label thatindicates that the contents are to be administered to a subjectresistant to amphetamines. In another embodiment, the kit can include asublingual formulation of a GABA_(A) receptor antagonist such asflumazenil and a label that indicates that the contents are to beadministered to a subject positive for increased production ofendozepines or other somnogenic compounds, as described herein. In afurther embodiment, a kit can include a sublingual formulation offlumazenil and a label that indicates that the contents are to beadministered with a wakefulness promoting agent and/or a transdermalformulation of flumazenil.

Also provided herein are kits for performing a diagnostic assay. In someembodiments, the diagnostic assay can be used to diagnose subjectssuffering from a GABA_(A) receptor mediated hypersomnia and/or todetermine subjects that would benefit from treatment with a GABA_(A)receptor antagonist. In some embodiments, a kit for use as a diagnosticassay is provided with the components for carrying out a patch clampassay as described herein. In some embodiments, the kit can include aGABA_(A) receptor antagonist and cells which transiently or stablyexpress human α1β2γ2s GABA_(A) receptors. In some embodiments, the kitcan include cells which transiently and stably express human α1β2γ2sGABA_(A) receptors and cells which transiently and stably express abenzodiazepine insensitive subunit (e.g., α1(H102R). In someembodiments, the kit further comprises one or more of an extracellularsolution that can function as a control sample, e.g., a control CSFsample; an intracellular solution; an extracellular medium, amotor-driven solution exchange device; and instructions for use of thekit.

VI. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications, and other publications are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

A “subject” can include both mammals and non-mammals. Mammals include,for example, humans; nonhuman primates, e.g. apes and monkeys; cattle;horses; sheep; rats; mice; pigs; and goats. Non mammals include, forexample, fish and birds.

The expression “effective amount”, when used to describe an amount ofcompound in a method, refers to the amount of a compound that achievesthe desired pharmacological effect or other effect, for example anamount that results in reduced sleepiness.

The terms “treating” and “treatment” mean causing a therapeuticallybeneficial effect, such as ameliorating existing symptoms, preventingadditional symptoms, ameliorating or preventing the underlying metaboliccauses of symptoms, postponing or preventing the further development ofa disorder and/or reducing the severity of symptoms that will or areexpected to develop.

EXAMPLES Example 1 Endozepine Modulation of GABA_(A)R Function

HEK293 cells transiently expressing human α1, β2, and γ2s subunits weresuperfused at 1 mL/min with an extracellular solution (ACSF) containing145 mm NaCl, 3 mm KCl, 1.5 mm CaCl₂, 1 mm MgCl₂, 6 mm d-glucose, and 10mm HEPES-NaOH adjusted to pH 7.4. Whole cell patch clamp recordings fromcells voltage clamped at −60 mV were made using the Multiclamp 700Bamplifier (Molecular Devices, Sunnyvale, Calif.). The resistance of thepatch pipette was 4-6 M when filled with intracellular solution (145 mmN-methyl-d-glucamine hydrochloride, 5 mm dipotassium ATP, 1.1 mm EGTA, 2mm MgCl₂, 5 mm HEPES-KOH, and 0.1 mm CaCl₂ adjusted to pH 7.2). Inaddition to the continuous bath perfusion with extracellular medium,solutions including AS99-CSF (as described below), GABA and/orflumazenil were applied rapidly to the cell by local perfusion using amotor-driven solution exchange device (Rapid Solution Changer RSC-160;Molecular Kinetics, Indianapolis, Ind.). Solutions were exchanged withinapproximately 50 ms. Laminar flow out of the rapid solution changer headwas achieved by driving all solutions at identical flow rates (1.0mL/min) via a multichannel infusion pump (KD Scientific, Holliston,Mass.). The solution changer was driven by protocols in the acquisitionprogram of pCLAMP version 9.2 (Molecular Devices, Sunnyvale, Calif.).AS-CSF was isolated from AS99, a patient experiencing hypersomnia. Thepatient also exhibited apparent resistance to amphetamine treatment. Allother compounds were obtained from the Sigma-Aldrich Co.

Results indicated that AS-CSF had no intrinsic GABA efficacy, but itenhanced the amplitude of response to EC₂₀ concentrations of GABA (seeFIG. 1a ). In a second experiment, 4 μM flumazenil was co-applied withAS-CSF. The flumazenil immediately reversed the enhancing effect of theAS-CSF (see FIG. 1b ).

Positive modulation of GABA_(A) receptor function by 100% or more isnormal for concentrations of general anesthetic drugs that wouldanesthetize a human. The results indicate that AS-CSF contains apositive allosteric modulator of GABA_(A) Receptor that would havepotent sedative effects in a human. The reversal of this effect byflumazenil suggests that the positive modulator likely acts directly orindirectly at the benzodiazepine binding site on the GABA_(A) receptor.

Accordingly, patients experiencing disorders associated with excessivesleepiness (e.g., idiopathic or amphetamine resistant hypersomnia) whotest positive for a positive allosteric modulator of GABA_(A) receptorfunction may likely benefit from administration of flumazenil.

Example 2 Formulation of Flumazenil as Tablet for Sublingual Dosing

Ingredient: Amount added: Flumazenil 0.3 grams Tablet triturate base(20%/80% powder) 4.7 grams Tablet triturate exipient (flavorless) 2milliliters Flavor, PCCA Bittershop 4 drops Stevia concentrate (250mg/mL) 2 drops

Procedure: The ingredients were combined and mixed to form a thickpaste. After the thick paste was formed, a flavor was added. The flavoradded was selected from the following:

-   -   a) 2 drops lemon, 1 drop marshmallow, 4 milligrams yellow color    -   b) 2 drops crème de mint, 4 mg green color    -   c) 2 drops tangerine, 1 drop marshmallow, 4 mg orange.        The formulation provided 50 tablets.

Example 3 Formulation of Flumazenil as Tablet Triturate for SublingualDosing

Ingredient: Amount added: Flumazenil 0.6 grams Tablet triturate base(20%/80% powder) 9.4 grams Tablet triturate exipent (flavorless) 4milliliters Flavor, PCCA Bittershop 8 drops Stevia concentrate solution(250 mg/mL) 4 drops

Procedure: The ingredients were combined and mixed to form a thickpaste. See Example 4 for tablet triturate base (20%/80% powder)formulation and Example 5 for stevia concentrate solution formulation.After the thick paste was formed, a flavor was added. The flavor addedwas selected from the following (quantities given are per 50 tablets):

-   -   a) 2 drops lemon, 1 drop marshmallow, 4 milligrams yellow color    -   b) 2 drops crème de mint, 4 mg green color    -   c) 2 drops tangerine, 1 drop marshmallow, 4 mg orange    -   d) 5 drops cherry, 2 drops vanilla, 4 mg red color.        The formulation provided 100 tablets.

Example 4 Formulation of Tablet Triturate Base 20%/80% Powder

Ingredient: Amount added: Sucrose powdered (confectioners) 20 gramsLactose monohydrate (hydrous) 80 grams

Procedure: The sucrose and lactose monohydrate were sieved through 120or smaller mesh. After adding the active ingredient (e.g., flumazenil),the mixture was wetted with an excipient of 40% distilled water and 60%alcohol. The formulation provided 100 grams of table triturate base20%/80% powder.

Example 5 Formulation of Stevia Concentrate Solution (250 mg/mL)

Ingredient: Amount added: Stevia powder extract 25 grams Sodium benzoate0.6 grams Water preserved liquid 100 milliliters

Procedure: The stevia powder and sodium benzoate were dissolved in thewater preserved. See Example 6 for water preserved liquid formulation.The mixture was warmed to aid in dissolution. The formulation prepared100 mL of stevia concentrate solution.

Example 6 Formulation of Water Preserved (Paraben) Liquid

Ingredient: Amount added: Water preserved concentrate liquid 10milliliters Water distilled liquid 3780 mL

Procedure: The liquids were mixed to prepare the water preserved(paraben) liquid. See Example 7 for water preserved concentrate liquidformulation.

Example 7 Formulation of Water Preserved Concentrate Liquid

Ingredient: Amount added: Methylparaben 19 grams Propylparaben NF 9.6grams Propylene glycol USP 100 mL

Procedure: The ingredients were mixed together and stirred until themethylparaben and propylparaben NF were completely dissolved.

Example 8 Formulation of Flumazenil as Cream for Transdermal Dosing

Ingredient: Amount added: Flumazenil 0.04 grams Prophlene glycol USP 0.1milligrams Food color, pink (powder) 0.03 milligrams Versabase cream 10grams

Procedure: The ingredients were combined and mixed. The formulationprovided 10 milliliters of cream.

Example 9 Formulation of Flumazenil as Cream for Transdermal Dosing

Ingredient: Amount added: Flumazenil 0.25 grams Prophlene glycol USP0.25 milliliters Food color, red (powder) 0.0075 milligrams Versabasecream 25 grams

Procedure: The ingredients were combined and mixed. The formulationprovided 25 milliliters of cream.

Example 10 Characterization of the Spectrum of GABA_(A) ReceptorMediated Hypersomnia

An organized, multidimensional approach to characterizing the phenotypicspectrum of GRH will be employed to determine who is affected, and howit manifests with specific attention to overlap with ICSD-2 definedsleep and circadian rhythm disorders. This will involve recruiting andextensively characterizing and correlating biological activity at theGABA_(A) receptor with behavior in 70 individuals suffering fromsleepiness or hypersomnia. Ten, age and sex-matched controls deemed‘affected’ or ‘unaffected’ by sleepiness will also be studied. Initialidentification, recruitment, and biological sample procurement will takeplace in the outpatient clinic and diagnostic sleep laboratory whichshare dedicated space. After satisfying inclusion/exclusion criteria andupon providing consent, additional behavioral, wake/sleep, andrest-activity cycle assessments will be conducted along withquantification endogenous GABA_(A) receptor bioactivity. Subjects willthen be admitted to a clinical setting for 24 hours and their clinicalresponse to single-blind intravenous delivery of saline, 0.5, and 2.0 mgflumazenil will be determined. Other known causes of hypersomnia, suchas hypocretin deficient narcolepsy, exogenous BZD use, iatrogeniceffects of common medications known to positively or negatively modulateGABA_(A)R (e.g., steroids, methylxanthines and many antibiotics), andmetabolic disorders (e.g., urea cycle disorders) will be excluded.Finally, to offer some further sense of the commonality and phenotypicspectrum associated with plasma potentiation of GABA_(A)R function, thisactivity will be quantified in a population-based sample of subjects.

Inclusion/Exclusion Criteria

Patients complaining of daytime sleepiness/hypersomnia with an EpworthSleepiness Scale or >15 and who exhibit either objective sleepiness(MSL<8 minutes), REM-sleep propensity during their diagnosticevaluation, or treatment resistant sleepiness will be recruited.Patients with DSM-IV Axis I disorders such as depression, bipolardisease, serious medical co-morbidities such as stroke, congestive heartfailure, active cancer, severe obstructive pulmonary disease, asthma, oruncontrolled type I or II diabetes will be excluded. Any patient with ahistory of CNS trauma, infection, or neurodegenerative condition will beexcluded. Patients with, treated or untreated sleep disordered breathing(AHI>10) will also be excluded. Subjects with chronic health conditionsotherwise well-controlled with medication (e.g., hypertension,hypothyroidism, arthritis) will be allowed to participate. Potentialcontrols and subjects will be excluded if they are ingestingpsychoactive medications including sedative-hypnotics, anxiolytics,mood-stabilizers presumed to act via GABAergic mechanisms, neuroleptics,and anti-depressants. In addition, given the known ability of steroids,methylxanthines, and many antibiotics to allosterically modulateGABA_(A)R, potential subjects taking gluco- or mineralo-corticoids,theophylline, or certain antibiotics will be excluded (at least whilethey are ingesting these agents). Three mls each of plasma and urinewill be sent to MedTox Laboratories (Burlington, N.C.) to be analyzedfor classic BZDs and their metabolites by gas chromatography (GC) andhigh performance liquid chromatography (HPLC). The specific agents andrespective reporting limits (i.e., thresholds for detection) willinclude: Desalkylflurazepan (flurazepam metabolite) 10 ng/ml;Nordiazepam 50 ng/ml; oxazepam 50 ng/ml, lorazepam 10 ng/ml, diazepam 50ng/ml, hydroxyflurazepam 10 ng/ml, temazepam 50 ng/ml, chordiazepoxide,50 ng/ml, midazolam 10 ng/ml, flurazepam 10 ng/ml,alpha-hydroxyalprazolam 50 ng/ml, alprozolam, 13 ng/ml, hydroxytriazolam10 ng/ml, triazolam 10 ng/ml and estazolam 10 ng/ml. Additional,individual samples will be sent to NMS Labs (Willow Grove, Pa.) for GCquantification of zolpidem (4-5 ng/ml), HPLC quantification for zaleplon(3 ng/ml), and HPLC tandem mass spectrometry (LC-MS/MS) quantificationof eszopiclone.

In order to more carefully delineate a provisional diagnosis of GRH andto provide additional potentially important biochemical data relevant tothe spectrum of hypersomnolence disorders such as narcolepsy withcataplexy, CSF for hypocretin (HCRT-1) will be assayed using acommercially available RIA (Orexin A RIA kit, Phoenix Pharmaceuticals,Belmont, Calif.). This assay has an intra-assay variability of <5%.Other recognized metabolic causes of hypersomnolence will also bescreened. For example, disorders of the urea cycle and the catabolicenzymes for GABA (e.g., GABA-transaminase and succinic semialdehydedehydrogenase) have been associated with lassitude and hypersomnia,albeit, incompletely characterized by MSLT or ICSD-2. These must beruled out as a potential contributors to hypersomnia by assessingarterial ammonia and urine and plasma organic and amino acids. Thelatter analyses will be performed in a CLIA certified laboratoryemploying ion exchange chromatography.

Flumazenil Infusion

The subject will be instructed in the proper use and care of theActiwatch and completion of the sleep/wake diary within one monthfollowing the lumbar puncture. The subject will complete all studyinventories (see below), and within two weeks, will undergo 48-hours ofambulatory polysomnography (see below). Five home/clinic visits will bemade during that period to hook-up the subject, check the integrity ofthe electrodes, and to disconnect the subject from the equipment. Withintwo weeks, subjects will be scheduled for a 24-hour admission to theACTSI and after a full-night of recorded sleep receive saline (control),0.5 mg, and 2.0 mg flumazenil at roughly 2.5 hour intervals whileundergoing continuous EEG monitoring and hourly monitoring of vitalsigns. All subjects will complete a baseline Stanford Sleepiness Scale(SSS) and Psychomotor Vigilance Task (PVT) (see below) which will berepeated at 10, 30, 60, 90, 120, and 150 minutes following eachinjection.

Polysomnographic Recording

Diagnostic nocturnal polysomnography (NPSG) and subsequent daytimetesting will b recorded with the Embla digital PSG system (MedcareCorporation, Buffalo, N.Y.) with a sampling rate of 512 Hz, as thisallows for Fast Fourier Transform of EEG signals. The system employs aWindows XP platform and uses proprietary software (Somnologica Science).Spectral analyses, or the Welch method of FFT smoothing, that provide anaverage of several FFT's will be useful to more fully characterize thesignature of fingerprint of endogenous GABA_(A)R like activity given theknown effects of GABA on corticothalamic excitability as manifest in theEEG.

Multiple Sleep Latency Testing

Daytime sleepiness will be objectively assessed with the MSLT, which isa clinical and research tool that uses standard guidelines for testingand scoring. Sleep latencies and number of REM onsets will be determinedaccording to standard criteria. The MSLT displays excellent interraterand intrarater reliabilities for sleep latency (coefficients of0.81-0.88) and REM onset scores (kappa coefficients of 0.78-0.88). Thestability of the MSL on repeat testing in known narcoleptics is high(r=0.81, p<0.01) with test-retest reliability improving vis a visdiagnostic certainty with the additional ICSD-2 requirement of two ormore sleep onset REM-sleep periods (Kappa=0.95; variance=0.08; Z=2.33;p<0.05).

Blood Collection

Thirty mL of venous blood will be drawn for: 1) lymphoblastoid cell linegeneration to establish a permanent source of DNA and cells for futureinvestigations; 2) clear plasma aliquoted and frozen for future analyticstudies; and 3) buffy coat and purified DNA banked for future geneticstudies. The DNA will be purified from 200 μl of buffy coat using aQiagen kit protocol (Qiagen, Valencia, Calif.). The cell lines, buffycoat, plasma and purified DNA will all be labeled with barcodecompatible labels and banked at −80° C. within CRIN dedicated resources.De-identified DNA from all participants will be assigned a 6-digitreference number. Aliquots with this reference number will be forwardedto a laboratory for testing.

Collection of Lumbar CSF

All patients and family members (afflicted, unafflicted) will provideInformed Consent for collection of cerebrospinal fluid (CSF). Lumbarpunctures (LP) will be performed under sterile conditions using standardprocedures, subcutaneous administration of 4% lidocaine, and collectionof 15-20 ml CSF with a 22 gauge spinal needle inserted at L3/L4 orL4/L5. One ml fractions will be labeled with the participants 6-digitreference number and frozen immediately upon dry ice and then stored at−80 degrees Centigrade for future analyses. LPs will be performedbetween 0830 and 0930 after completion of the first MSLT nap. This willobviate the need to control for subsequent daytime activity levels andextent of food intake which hypothetically could affect endogenousactivity at the GABA_(A)R.

Questionnaire Assessments

Administered questionnaires serve as both screening instruments and aspredictors in the regression models described below. Subjectivesleepiness as a trait variable will be assessed using the EpworthSleepiness Scale (ESS) and overall quality of sleep will be assessedusing the Pittsburgh Sleep Quality Index (PSQI). State and trait anxietywill be assessed with the State-Trait Anxiety Inventory (STAI) and moodwill be assessed with the Beck Depression Inventory (BDI). These are allstandardized scales with population based norms. Data on functionalimpairments related to sleepiness using the Functional Outcomes of SleepQuestionnaire (FOSQ) will also be collected. The FOSQ is a self-reportmeasure designed to assess the impact of excessive sleepiness onmultiple activities of daily living.

Actigraphy

The Actiwatch wrist-worn monitor, manufactured by Respironics(Murrysville, Pa.), will be used to assess characteristic sleepdurations in patients for two weeks prior the infusion protocol.Patients will also be provided a sleep log to keep during the two weeksto generate data on timing of sleep and napping.

Ambulatory Polysomnography

Ambulatory PSG over a 48 hour period will be conducted using the sameequipment cited above which can be adapted for this use to document thedegree of ‘hypersomnia’ suggested by actigraphy. Only EEG, EOG,submental EMG lead, ECG, and pulse oxymetry will be conducted. No limbleads will be used for patient safety reasons. Sleep stages, episodes ofdesaturation, and ECG will be analyzed.

Psychomotor Vigilance Task (PVT)

The Psychomotor Vigilance Task (PVT) provides a sensitive marker ofminute-to-minute fluctuations in alertness during the flumazenilinfusion protocol. The PVT is a 10-minute, simple, portable reactiontime test (finger button press response to light) designed to evaluatethe ability to sustain attention and respond in a timely manner tosalient signals. Data to be generated include: 1) frequency of lapses,which refer to the number of times the subjects fail to respond to thesignal or fail to respond in a timely manner; 2) the median reactiontime (RT) over the 10-minute interval. Additionally, as a measure ofstate sleepiness, the Stanford Sleepiness Scale (SSS) will beadministered immediately prior to each trial. The PVT/SSS will beadministered at 10, 30, 60, 90, 120, and 150 minutes following eachinfusion of saline or flumazenil.

Statistical Analysis/Power Calculations

The relationship between the extent of GABA_(A) potentiation andbehavioral outcomes will be examined using regression models. Separatemodels will be run for each type of specimen source (e.g., CSF andplasma derived markers of potentiation). A simple bivariatc relationshipbetween the two measures of GABA potentiation using correlational modelswill be examined, relying on non-parametric alternatives (Spearman)should the measure present with a non-normal distribution. The extent ofGABA potentiation in the Baseline condition among patients will bepredicted using predictors such as standard demographics (e.g., gender,age), psychometrics (e.g., STAI, BDI), recent sleep history (e.g.,cumulative sleep over the preceding 2 weeks as measured with actigraphy,daytime naps on sleep log), and laboratory-based measurements ofnocturnal sleep (e.g., FFT derived relative delta power or beta power)or daytime alertness (e.g., MSLT sleep latency, PVT-derived medianreaction time). Because multiple measurements in each domain and thesheer number of domains increase the likelihood of Type I error, sucherror will be minimized by first carefully examining theintercorrelations among measures within each domain. Substantialcollinearity is expected to be among many of these. For example, traitanxiety (STAI) and depressed mood (BDI) are likely to be highlyintercorrelated, as are Baseline PVT median reaction times andMSLT-defined sleep latency. The specific approach to deriving variablesto employ in the regression might include a selection of a singlevariable from each domain chosen on the basis of a more normallydistributed range of scores across subjects. Alternatively, the datareduction techniques can be relied on such as principal componentsanalyses (PCA) to determine a single measure in each domain that bestcaptures variance within that domain. Thus, a single score (or compositescore, if PCA was used) from each domain will be entered in theregression predicting potentiation. Based on the data presented in FIG.3, large effects will be displayed. GABA potentiation differencesbetween controls and patients will be substantial (d=3.095). Assumingeffects of this size are maintained in the work proposed here, andassuming a 2-tailed alpha of 0.01, an N of 60 cases would yield 99%power to reject the null of hypothesis of the contribution for anysingle domain to GABA potentiation. It is fully recognized that, inmultivariate models encompassing each of the five domains listed above,actual power might be somewhat reduced because of the contribution ofmultiple variables to the prediction. Nonetheless, given the substantialeffects observed in FIG. 3, sufficient power to understand how differentvariables may predict potentiation when considered simultaneously shouldbe retained. Regression models will also be used to determine whatfactors may predict change in GABA potentiation under flumazenilinfusion. Each patient's Baseline potentiation level (measured undersaline infusion) will be forced and it is determined whether either lowor high dosage of flumazenil predicts change subsequent to infusion.Domain variables selected for entry into these models are limited onlyto those shown to relevant to the prediction of Baseline potentiation,thus saving degrees of freedom whenever possible. This modeling allowsthe determining of the extent that other variables (demographic, recentsleep history, etc) may have to moderate or mediate the GABA-mediatedresponse to flumazenil. The behavioral response to flumazenil (performedseparately by dose) will also be examined, defined as the mean of themedian RTs for the 4 PVT measurements closest to point of infusion. Eachpatient's Baseline median RT (mean of 4 Baseline/saline measurements)(see FIGS. 6, 8, 10, and 12) and Baseline GABA potentiation levels willbe forced initially in these regressions, followed by entry ofsignificant predictors of Baseline potentiation found in the analysesdescribed above.

Further, the plasma-measured GABA potentiation will be examined as thedependent variable among 227 individuals, all of whom will have receivedtwo nights of PSG and an intervening day of MSLT. The hypersomnolencedemonstrated by the index cases will represent a more extreme form of acontinuous trait present in segment of the population generally. To thatend, the initial review of the data indicated that 58 had meanMSLT-defined sleep latencies of less than 5 minutes that could not beaccounted for by known sleep disorders. If the MSLTs across all 227cases show a bimodal distribution, the analyses would be limited to onlythose cases at the extremes (e.g., mean latencies <5 minutes versus meanlatencies >15 minutes) and employing an ANCOVA approach. However, thedistribution of mean sleep latencies is more continuous and, as is oftenthe case of studies using MSLT, sharply skewed to the right. In thiscase, log transforms are performed on these mean values beforeproceeding. The overall approach will be similar to those describedabove, though they are somewhat more limited by the range of variablescollected.

Example 11 Electroencephalography (EEG) Power Spectrum Analysis

Quantitative analysis of delta (0.4-3.99 Hz), theta (4.00-7.99 Hz),alpha (8.00-12.99 Hz) and beta power (13.00-16.00 Hz) was obtained fromEEG spectral analyses of the C4-M1 electrode. Manual and automatedartifact removal methods were utilized prior to EEG spectral analyses toprevent erroneous results. Spectral analyses were conducted utilizingthe computational software program MATLAB v 7.1. The Welch method of FFTsmoothing was employed to obtain power spectrum values from an averageof several FFT's. The FFT contained a minimum of 512 data samples with a50% overlap moving window of the subsequent 512 data samples. EEG datacollected at 200 Hz provided an FFT window comprised of approximately2.56 seconds of data. Parameters for Welch spectral analyses were useradjustable within the MATLAB program such that user defined frequencybands for specific frequency resolutions were obtained. Power values forthe defined frequency bands were represented by mV²/Hz (microvoltssquared divided by hertz).

The EEG power spectrum analyses for two subjects (DS122 and DT74) wereobtained (see FIGS. 4 and 5, respectively). Table 1 provides thecorresponding sampling frequency and FFT window size for each patientdata set. There was a spectral change approximately five minutes afterintravenous infusion of 2.0 mg flumazenil that manifested as diminutionof delta frequencies and emergence of higher EEG frequencies emblematicof improved vigilance/arousal.

TABLE 1 Patient Sampling Frequency FFT size DS 500 Hz 1024 DT 200 Hz 512

Table 2 displays mean relative band power results obtained from EEGpower spectrum (i.e., delta, theta, alpha, beta) analyses of subjectED102 and DS122 for each clinical treatment (i.e., saline, 0.5 mgflumazenil, and 2.0 mg flumazenil). Ten minute data segments wereselected 30 minutes following each clinical treatment and were analyzedvia a three second processing window to obtain the relative powerspectrum results provided in Table 2.

TABLE 2 Delta Theta Alpha Beta Gamma Subject Treatment Power Power PowerPower Power ED102 Saline 0.5314 0.1824 0.1090 0.1207 0.0565 ED102 0.5 mg0.5199 0.1755 0.1017 0.1387 0.0642 flumazenil ED102 2.0 mg 0.4820 0.14790.0866 0.1803 0.1032 flumazenil DS122 Saline 0.4122 0.2777 0.1966 0.10110.0124 DS122 0.5 mg 0.3515 0.2995 0.2183 0.1165 0.0142 flumazenil DS1222.0 mg 0.3128 0.3776 0.1798 0.1141 0.0157 flumazenil

Example 12 Psychomotor Vigilance Task (PVT)

The dose and temporal reversibility of the sleepiness of patients tointravenous flumazenil were determined employing the PVT/SSS paradigm asdescribed in Example 10. Five hypersomnic patients demonstrateddose-dependent improvements in vigilance and subjective alertness withintravenous delivery of flumazenil as shown in Table 3.

TABLE 3 Baseline/Saline Stanford % Reaction Sleepiness FLU 0.325-0.5 mgFLU 1.2-2 mg GABA time (RT) Scale RTs in RTs in Case potentiation in mslapses (SSS) ms lapses SSS ms lapses SSS 74 200 +/− 21.7 432.3 +/− 84.831.0 6 236.8 +/− 47.7 0.7 4   207 +/− 4.97 0.7 3 99 160 +/− 9.2  285.5+/− 13 N/A 6 255.8 +/− 6.3 N/A 3 225.5 +/− 2.3 N/A 1 102 189 +/− 24.3 1962 +/− 1478 16.4 6  1642 +/− 1036 3.1 3 363.2 +/− 38.2 3.8 4 122 149+/− 20.4 369.5 +/− 78.9 17.2 5 297.8 +/− 16.2 0.6 1 269.3 +/− 6.8 0.6 1124 58.5 +/− 3.5   327.8 +/− 22.96  5.8 6 259.8 +/− 3.8 1.3 2   250 +/−1.2 1.0 2

Administration of flumazenil (FLU) was associated with dramatic andsubstantial improvement in reaction time performance on the PVT andsubjective alertness on the SSS. Relative to baseline, median RTsdecreases at low (t=2.56, p=0.063) dose, and number of lapses decreasedboth at low (t=3.03, p=0.056) and high (t=3.51, p=0.039) dose. Whencompared to the worst Baseline measure for each case, SSS showedsignificant improvement for both low (t=8.55, p=0.001) and high (t=7.06,p=0.002) dose. Raw histograms for cases 74, 102, 122, and 124 displayingbaseline PVT performance and PVT performance after 2.0 mg are shown inFIGS. 6-17.

Example 13 Clinical Study of GABA_(A) Receptor Mediated Hypersomnia(GRH)

Patient AS99 with a diagnosis of “narcolepsy” and restless legs syndrome(RLS) complained of “craving” sleep, and of long, unrefreshing sleepperiods. Polysomnography revealed periodic leg movements (31 per hour),but was otherwise normal (TST=444 min). A mean sleep nap latency of 2.6minutes absent intrusion of REM sleep confirmed pathological sleepinessand a diagnosis of idiopathic hypersomnia. Patient AS99's examinationwas normal with a BMI of 22.3, and urine drug screens (repeated×3),serum ammonia (n=2), thyroid functions (n=2), complete blood counts(n=5), vitamin B12, and comprehensive metabolic screens (n=2) werenormal. Ferritin (23 ng/ml) and % transferrin saturation (13%) were lowwith otherwise normal serum iron. The RLS was successfully treated withiron supplementation and pramipexole; however, hypersomnia persisteddespite maximum doses of dextroamphetamine (60 mg) in combination withmodafinil (800 mg). Actigraphy confirmed resolution of RLS/PLMs, yetrevealed erratic rest-activity cycles with sleep periods varying from 5to 10 hours per night. Patient AS99's condition progressed and weightdecreased (BMI=20), and patient AS99 developed anxiety and hypertensionrequiring treatment with metoprolol attributed to supratherapeutic dosesof psychostimulants. Affective and factitious disorders were ruled outby two independent psychiatric assessments. Weaned off all medications,CSF was obtained and hypocretin determined to be high-normal (401 pg/ml)thus ruling out a diagnosis of narcolepsy. Electrophysiological analysisfor bioactivity in CSF and plasma revealed the presence of a positiveallosteric modulator of the GABA_(A) receptor reversible with thecompetitive BZD antagonist flumazenil. The dose and temporalreversibility of sleepiness to intravenous flumazenil were thendetermined employing the PVT/SSS paradigm. The rest-activity cycles ofpatient AS99 improved with chronic sublingual flumazenil administration(see FIGS. 18a and 18b ). The sleep, mood, sleepiness, and quality oflife improved dramatically and are sustainable with sublingualflumazenil in patient AS99 (see Table 4) as shown through the PittsburghSleep Quality Index, Beck Depression Inventory, Epworth SleepinessScale, Functional Outcomes of Sleep, and SF-36 Health Survey.

TABLE 4 February 22^(nd) March 31^(st) (1 April 28^(th) Variable(pre-treatment) month post) (2 months post) Pittsburgh Sleep 4 2 1Quality Index Beck Depression 7 2 2 Inventory Epworth Sleepiness Scale18 3 3 Functional Outcomes of Sleep General Productivity 6 23 24 SocialActivity 12 24 24 Activity 3.6 19.6 21.3 Vigilance 8 22 24 Total 29.588.6 93.3 Total Mean 7.4 22.1 23.3 SF-36 Health Survey Physical 39.554.4 66.2 Mental 49.7 64.0 56.2

Patient AS99 continued use of sublingual flumazenil for 9 months withpositive results. When prescribed clarithromycin, patient AS99 suddenlydeveloped 4 nights of insomnia that reversed promptly upondiscontinuation. Clarithromycin is an antibiotic with a high incidenceof hypomania/insomnia associated with its use, and it functions as anegative allosteric modulator at GABA_(A) receptors.

Example 14 Identification of Substance Causing Potentiation at GABA_(A)Receptors

Several studies were performed in order to identify the substanceaccounting for potentiation at GABA_(A) receptors. It was determinedthat adenosine is not the substance, as several concentrations (1 mM,100 μM, and 10 μM) of adenosine in artificial CSF exhibited no activityat GABA_(A) receptors.

In addition, it was shown that the substance accounting for potentiationat GABA_(A) receptors is not a neurosteroid. Cerebrospinal fluid fromfour hypersomnic cases were tested in duplicate by quantitative HPLC forendogenous neuroactive GABAergic steroids (i.e., neurosteroids). Thecontrols revealed no differences in the levels of pregnenolone, DHEA,3α,5α-THP, 3α,5β-androstandiol, 3α,5α-androsterone, and3α,51β-androsterone. Controls and subjects exhibited undetectablequantities of 3α,5β-THP, 3α,5α-THDOC, 3α,5β-THDOC, and3α,5α-androstandiol.

Further, it was shown that the substance accounting for potentiation atGABA_(A) receptors has a molecular weight less than 3,000. Pooled CSFsfrom confirmed GRH subjects versus controls were fractionated withfilters having approximately 3,000 molecular weight cut-off. Bioactivityat GABA_(A) receptors in both samples was completely retained within thesmaller molecular weight fractions.

It was also found that the substance accounting for potentiation atGABA_(A) receptors may act at a non-traditional benzodiazepine site (seeFIGS. 19a and 19b ). In a whole cell patch clamp current recording froma cell expressing human α1β2γ2s receptors, the response to 10 μM GABA ispotentiated by the co-application of a 50% CSF, indicating the presenceof a positive allosteric modulator (see FIG. 19a ). A recording from adifferent cell expressing the benzodiazepine insensitive subunitα1(H102R) shows that the enhancement persists (see FIG. 19b ). Not to bebound by theory, this data indicates that the somnogenic compound is nota classical benzodiazepine, or does not act conventionally at theclassical high-affinity benzodiazepine binding site on the GABA_(A)receptor.

Example 15 Patch Clamp Analysis of CSF Bioactivity

Patch clamp analyses of CSFs from non-human primates, drawn from animalsunder different conditions, was also used to identify the somnogenicGABAergic substance. In this experiment, CSF was drawn from 4 monkeys(Canjala, Santiaga, Penelope, and Cricket) at 3 different timepoints. 1) early morning, 2) late afternoon and 3) very late evening,having been kept awake throughout (when they would normally be asleep).A whole cell patch clamp current was recorded (as described in Example1), and the response to 10 μM GABA co-administered with primate CSF wasdetermined. The bioactivity of the CSF is expressed as a percentincrease, or potentiation, of the control current by the CSF (see Table5). The first two columns show the normal diurnal variation of thissomnogenic compound. It appears that this substance waxes and wanes inanimals as it does in humans during the normal day night cycle. Moreinterestingly, in 2 of the 4 animals, the bioactivity increased stillfurther after the animals were “wake-extended”. These promising resultsindicate that under sleep deprived conditions, humans may also benefitfrom flumzenil or other GABAergic therapy to relieve the symptoms offatigue they experience from the accumulation of this somnogeniccompound.

TABLE 5 Morning Evening Wake Enhanced Canjala 79.9 ± 2.7 76.6 ± 4.6 73.4± 4.7 Santiaga 49.0 ± 2.0 58.7 ± 0.8 64.9 ± 1.7 Penelope 54.6 ± 3.5 77.1± 0.5  98.4 ± 23.1 Cricket 51.3 ± 0.7 58.1 ± 4.2 63.9 ± 6.9

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating hypersomnia, the methodcomprising administering an effective amount of a GABA_(A) receptorantagonist to a subject having hypersomnia, wherein the GABA_(A)receptor antagonist is administered transmucosally or transdermally, andwherein the GABA_(A) receptor antagonist is selected from the groupconsisting of flumazenil, clarithromycin, picrotoxin, bicuculline,cicutoxin, and oenanthotoxin.
 2. The method of claim 1, wherein theGABA_(A) receptor antagonist is flumazenil or clarithromycin.
 3. Themethod of claim 1, wherein the transmucosal administration of theGABA_(A) receptor antagonist is combined with the transdermaladministration of the GABA_(A) receptor antagonist.
 4. The method ofclaim 1, wherein the GABA_(A) receptor antagonist is administeredtransmucosally in a tablet, packaged powder, edible film strip, soft gelcapsule, hard gel capsule, lozenge, or troche.
 5. The method of claim 1,wherein the GABA_(A) receptor antagonist is administered transmucosallyin a unit dosage form comprising from about 0.5 mg to about 20 mg of theGABA_(A) receptor antagonist.
 6. The method of claim 5, wherein theGABA_(A) receptor antagonist is administered transmucosally in a unitdosage form comprising about 6 mg of the GABA_(A) receptor antagonist.7. The method of claim 1, wherein the GABA_(A) receptor antagonist isadministered transmucosally at a daily dosage from about 1 mg per BMI toabout 5 mg per BMI.
 8. The method of claim 1, wherein the GABA_(A)receptor antagonist is administered transdermally in an ointment, anemulsion, a lotion, a solution, a cream, a gel, or a patch.
 9. Themethod of claim 1, wherein the GABA_(A) receptor antagonist isadministered as a daily dose.
 10. The method of claim 9, wherein theGABA_(A) receptor antagonist daily dose is divided equally into two tosix times per day daily dosing.
 11. The method of claim 1, wherein thesubject was not administered a benzodiazepine.
 12. The method of claim1, wherein the subject was not administered midazolam.
 13. The method ofclaim 1, wherein the subject has a measured Epworth sleepiness scale ofgreater than
 15. 14. A method of treating hypersomnia in a humansubject, the method comprising administering an effective amount of aGABA_(A) receptor antagonist to the human subject, wherein the GABA_(A)receptor antagonist is administered transdermally, transmucosally,sublingually, or subdermally, wherein the GABA_(A) receptor antagonistis selected from the group consisting of flumazenil, clarithromycin,picrotoxin, bicuculline, cicutoxin, and oenanthotoxin.
 15. The method ofclaim 14, wherein the GABA_(A) receptor antagonist is flumazenil orclarithromycin.
 16. The method of claim 14, wherein the subject hadhypersomnia over the course of a year or more prior to beingadministered with the GABA_(A) receptor antagonist.
 17. The method ofclaim 14, wherein a cerebrospinal fluid (CSF) or blood sample from thesubject having hypersomnia potentiates the response of GABA onGABA-induced currents in a cell expressing a GABA_(A) receptor.
 18. Themethod of claim 14, wherein the GABA-induced currents in the cellexpressing the GABA_(A) receptor are measured by a whole cell patchclamp.
 19. The method of claim 14, wherein the expressed GABA_(A)receptor is a recombinant human GABA_(A) receptor.